title: Detection of Ehrlichia canis and Anaplasma platys DNA using multiplex PCR.
abstract: We hereby propose a novel sensitive, specific, and cost-efficient method to detect Ehrlichia canis and Anaplasma platys DNA from canine whole blood samples by multiplex PCR. Blood samples from hemoparasited dogs attending the Veterinary Hospital at the Universidade Federal Rural da Amazônia-UFRA, Belém, Brazil, were collected in tubes containing EDTA. Amplification of E. canis and A. platys 16S rDNA by nested (n) PCR was successfully achieved by using primers specific to the Anaplasmataceae in the first round of PCR, followed by a second round of PCR using E. canis-specific primers in conjunction with A. platys-specific primers. The amplicons obtained were cloned and sequenced, yielding sequences of 478 and 473 bp (including primers) pertaining to regions of the 16S rDNA of E. canis and A. platys, respectively. The protocol we here propose may help to measure the prevalence of canine monocytic ehrlichiosis (CME) and canine cyclic thrompocytopenia, not only in northern Brazil, where there is no data available, but also elsewhere.
authors.affiliation: 1 Laboratório de Tecnologia Biomolecular, Instituto de Ciências Biológicas, Universidade Federal do Pará , Cidade Universitária, Belém, Pará, Brazil .
authors.forename: Claudia Pinheiro
authors.initials: CP
authors.lastname: Rufino
authors.forename: Pablo Henrique Gonçalves
authors.initials: PH
authors.lastname: Moraes
authors.forename: Thais
authors.initials: T
authors.lastname: Reis
authors.forename: Ruan
authors.initials: R
authors.lastname: Campos
authors.forename: Délia Cristina Figueira
authors.initials: DC
authors.lastname: Aguiar
authors.forename: John Anthony
authors.initials: JA
authors.lastname: McCulloch
authors.forename: Andre Marcelo Conceição
authors.initials: AM
authors.lastname: Meneses
authors.forename: Evonnildo Costa
authors.initials: EC
authors.lastname: Gonçalves
mesh_terms: D000709:Anaplasma / Q000235:genetics / Q000302:isolation & purification*; D000712:Anaplasmosis / Q000175:diagnosis* / Q000453:epidemiology; D000818:Animals; D001483:Base Sequence; D001938:Brazil / Q000453:epidemiology; D003362:Cost-Benefit Analysis; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000097:blood* / Q000737:chemistry / Q000235:genetics; D004285:Dogs; D041061:Ehrlichia canis / Q000235:genetics / Q000302:isolation & purification*; D016873:Ehrlichiosis / Q000175:diagnosis* / Q000453:epidemiology; D005260:Female; D008297:Male; D008969:Molecular Sequence Data; D060885:Multiplex Polymerase Chain Reaction / Q000191:economics / Q000379:methods* / Q000592:standards; D015995:Prevalence; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA / Q000662:veterinary; D013997:Time Factors
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S